Inorganic phosphates (hereinafter, may be referred to as “phosphate”) are essential in energy metabolism in vivo and maintenance of cellular functions, and play an important role in tissue calcification in cooperation with calcium. Supply of phosphate to an organism depends mainly on absorption in the intestinal tract, and phosphate excretion depends on urinary excretion in the kidney and fecal excretion in the intestinal tract. In living organisms, phosphate is distributed in body fluid, intracellular fractions and calcified tissues. The level of excretion of inorganic phosphate in an adult is maintained at almost the same level of absorption of inorganic phosphate, suggesting the presence of a regulatory mechanism which maintains homeostasis of the phosphate metabolism. It is known that the metabolism of calcium, which shares similarity with the phosphate metabolism in terms of a distribution and homeostatic control of blood level, is controlled in a co-operative manner in mammals by regulatory factors, such as, at least parathyroid hormone, calcitonin and 1α,25-dihydroxyvitamin D3.
In the regulation of phosphate metabolism it is known that parathyroid hormone promotes phosphate excretion, and that 1α,25-dihydroxyvitamin D3 promotes phosphate absorption in the intestinal tract. This clearly suggests close association between phosphate metabolism and calcium metabolism. However, a substance primarily controlling phosphate has not yet been elucidated.
Now, examples of a disease which is associated with the loss of the homeostasis of phosphate metabolism and lower inorganic phosphate levels in the blood include primary hyperparathyroidism, hereditary hypophosphatemic rickets, and tumor-induced osteomalacia.
Primary hyperparathyroidism is a disease characterized by an overproduction of parathyroid hormone in the parathyroid glands, and is known to develop hypophosphatemia with increased phosphate excretion because overproduced parathyroid hormone suppresses reabsorption of inorganic phosphate in the kidney.
Further, known examples of hypophosphatemia resulting from hereditary diseases include type I vitamin D-dependent rickets, type II vitamin D-dependent rickets and vitamin D-resistant rickets. Type I vitamin D-dependent rickets is a disease caused by hereditary dysfunction of the synthase to produce active vitamin D metabolites, and type II vitamin D-dependent rickets is a disease caused by hereditary dysfunction of vitamin D receptor. Both diseases develop hypophosphatemia together with hypocalcemia due to attenuated action of vitamin D3 metabolites. In contrast, for vitamin D-resistant rickets, at least 2 types of clinical conditions, X-linked chromosomal and autosomal hypophosphatemic rickets resulting from different causes are known to exist.
Both of the above-mentioned clinical conditions of vitamin D-resistant rickets lead to hypophosphatemia characterized by renal phosphate. Recently, it has been shown in patients with X-linked hypophosphatemic rickets (hereinafter, also referred to as “XLH”) that the disease is induced by mutations in the gene encoding an endopeptidase-like protein, named PHEX, on X chromosome. However, a mechanism how dysfunction of PHEX protein induces hypophosphatemia has not been elucidated. Interestingly, gene analysis of a naturally occurring mutant mouse (Hyp) which developed hypophosphatemia has revealed the partial deletion of the gene encoding PHEX in this mouse. Experiments using these mice have revealed that PHEX deficient mice have normal renal function, and a humoral factor, which is different from parathyroid hormone, but induces hypophosphatemia, is present in the body fluid of Hyp mice. Concerning autosomal dominant hypophosphatemic rickets/osteomalacia (hereinafter also referred to as ADHR), a gene responsible for this disease has been pursued, and the presence of such a gene in 12p13 region has been indicated by linkage analysis. However, the region that has been narrowed down so far is still wide and contains many genes, so that no candidate gene has been specified yet.
Tumor-induced osteomalacia is a disease which develops hypophosphatemia with increased renal phosphate in association with tumorigenesis, and is characterized in that the hypophosphatemia is eliminated by irradiation to tumor or removal of tumor. In this disease, it is thought that tumor produces a factor which induces hypophosphatemia due to suppressed reabsorption of phosphate in the kidneys.
It has not been confirmed whether a putative causative molecule for vitamin D-resistant rickets is identical to that for tumor-induced osteomalacia. However, the two factors are identical in that they clearly are unknown phosphate metabolism factors which promote urinary phosphate excretion. The putative phosphate metabolism regulatory factor is often referred to as, the name Phosphatonin. The relationship of this unknown phosphate metabolism regulatory factor and vitamin D-resistant rickets or tumor-induced osteomalacia has been summarized as general remarks (Neison, A. E., Clinical Endocrinology, 47:635-642, 1997; Drezner, M. K., Kidney Int., 57:9-18, 2000).
Another characteristic of vitamin D-resistant rickets or tumor-induced osteomalacia is impairment of bone calcification. This impaired bone calcification could be thought to be secondarily developed by hypophosphatemia. However, since abnormal bone calcification in experiments using Hyp mice, the model mice for vitamin D-resistant rickets is shown to develop independently from phosphate levels (Ecarot, B., J. Bone Miner. Res., 7:215-220, 1992; Xiao, Z. S., Am. J. Physiol., E700-E708, 1998), it is conceivable that the above unknown regulatory factor for phosphate metabolism can directly regulate calcification in bone tissue.
As described above, research data have strongly been suggesting the presence of an unknown factor which regulates phosphate metabolism, but there has been no case that can elucidate at a molecular level, an entity which exhibits the putative activity. While WO99/60017 discloses a novel polypeptide sequence as a novel polypeptide hormone, Phosphatonin, however, it does not disclose the characteristic activity of phosphatonin which concerns induction of hypophosphatemia. Thus, it is conceivable that an unidentified intrinsic factor regulating phosphate metabolism may exist in organisms.
Vitamin D2 and vitamin D3 ingested from foods, or vitamin D3 synthesized in the skin is hydrolyzed by vitamin D-25-hydroxylase existing mainly in the liver to produce 25-hydroxyvitamin D. Then, 25-hydroxyvitamin D is hydrolyzed by 25-hydroxyvitamin D-1α-hydroxylase existing in renal epithelial cells of proximal tubules in the kidney to produce 1α25-dihydroxyvitamin D. This 1α,25-dihydroxyvitamin D is a mineral regulatory hormone having physiological activities that increase serum calcium and phosphate levels, and is known to be responsible for inhibiting the secretion of parathyroid hormone and to be involved in the promotion of bone resorption. 1α,25-dihydroxyvitamin D is then converted into metabolites in vivo which has not the above physiological activities by 24-hydroxylase existing mainly in the kidney or small intestine. In this regard, 24-hydroxylase is thought to be an enzyme which is responsible for the inactivation of 1α,25-dihydroxyvitamin D. On the other hand, 24-hydroxylase is known to also act on 25-hydroxy vitamin D and convert it into 24,25-dihydroxyvitamin D. The 24,25-dihydroxyvitamin D has been reported to have physiological effects that increase bone mass or promote differentiation of cartilage, suggesting that this enzyme has an aspect for generating biological active vitamin D metabolites.
Known factors that regulate the expression level of 1α-hydroxylase, which has an important role in the activation of vitamin D, include parathyroid hormone (PTH), calcitonin, 1α,25-dihydroxyvitamin D and the like. PTH whose secretion is promoted by decreases in blood calcium levels acts on PTH receptors existing in epithelial cells of the renal proximal tubules to promote transcription of 1α-hydroxylase gene through an elevated intracellular cAMP level, so as to increase blood 1α,25-dihydroxyvitamin D concentration. 1α,25-dihydroxyvitamin D promotes absorption of calcium from the intestinal tract and calcium reabsorption in the kidney, thereby increasing the blood calcium level. Further, it has been reported that the binding of 1α,25-dihydroxyvitamin D to vitamin D receptor (VDR) acts on a promoter region of 1α-hydroxylase gene or PTH gene to suppress the transcription of such genes. Specifically, 1α,25-dihydroxyvitamin D has a feedback control mechanism for its activation factor, PTH and 1α-hydroxylase. This mechanism plays an important role in maintaining homeostasis of calcium metabolism.
Recently, it has been reported that a decrease in serum phosphate level enhances the expression of 1α-hydroxylase gene. In phosphate metabolism, the presence of a mechanism is also assumed that enhancement in the expression of 1α-hydroxylase gene association with decreased serum phosphate level elevates serum 1α,25-dihydroxyvitamin D level and, consequently, corrects the serum phosphate level by promoting absorption of phosphate from the small intestine.
Examples of a factor responsible for regulating the expression of 24-hydroxylase gene include 1α,25-dihydroxyvitamin D and PTH. It has been shown that 1α,25-dihydroxyvitamin D interact with the vitamin D receptor (VDR) and the complex binds to a vitamin D receptor response sequence existing in the promoter region of 24-hydroxylase gene so as to promote transcription. 1α,25-dihydroxyvitamin D is thought to activate 24-hydroxylase, and then to induce a decrease in the 1α,25-dihydroxyvitamin D level due to the activated catabolic pathway. It is known that the expression of 24-hydroxylase gene is suppressed by PTH, but its detailed molecular mechanism is unknown.